This project aims to examine the efficacy of targeting amino acid transporters ASCT2 and LAT1 in human hepatocellular carcinoma (HCC), a recalcitrant cancer with poor prognosis and limited treatment options. ASCT2 and LAT1 are coordinately upregulated in a broad spectrum of primary human cancers, including HCC, but it is presently unclear what makes these two transporters (out of several) so coveted by malignancies. Neither transporter is expressed in normal liver cells (hepatocytes) at detectable levels making them attractive prospective targets for therapy. Moreover, ASCT2 and LAT1 have been shown to physically associate in the plasma membrane of human cancer cells, suggesting that they cooperate to help drive cancerous growth. Previous work from our laboratory showed that targeted silencing of ASCT2 elicits programmed cell death (apoptosis) in human HCC cells;here, we include LAT1 in the repertoire of transporter-targeted therapy. The primary objective of the currently proposed project is to test the broad applicability of targeted transporter therapy in HCC and in doing so, to assess the role of ASCT2 and LAT1 in HCC growth and survival signaling. This aim will be achieved through the use of 10 human hepatoma cell lines representing a broad clinically relevant spectrum of different HCC phenotypes. A stably maintained inducible expression system for short hairpin RNA (shRNA) targeting ASCT2 or LAT1 will be established in each of the 10 hepatoma lines, and utilized to assess the efficacy of targeting these transporters via RNA interference (RNAi) based silencing. The consequences of transporter silencing on growth and survival signaling (mTOR and Akt pathways, respectively) will be assessed through western blotting analysis. A second aim of the project will be to establish whether ASCT2 and LAT1 physically associate in the plasma membrane of HCC cells, and how silencing of one transporter affects the expression and activity of the other. The third aim of the project involves elucidating which cues present in the tumor microenvironment (amino acid limitation, hypoxia and acid pH) induce trafficking of the transporter complex to the plasma membrane where they function. In cases where transporter silencing does not induce apoptosis, their role in initial avascular growth (nascent tumor formation) will be assessed via spheroid formation (3-dimensional aggregation and growth of HCC cell lines). Collectively, these aims will give insights into the potential for developing transporter-targeted therapy, and the role of each in human HCC biology. PUBLIC HEALTH RELEVANCE: This project aims to investigate the potential of targeting two amino acid transporters upregulated in cancer ASCT2 and LAT1 as therapy for human hepatocellular carcinoma (HCC), and to determine their role in the development and growth of hepatocellular cancer. The incidence of HCC is on the rise in the United States due primarily to the increasing prevalence of chronic hepatitis C infection, and there are currently limited treatment options for this recalcitrant malignancy. Finding an "Achilles heel" in this cancer that is amenable to therapy is therefore paramount.